Blood is the circulating liquid that transports oxygen and nutritive materials to the tissues of the body and removes carbon dioxide and other metabolic products and wastes for excretion. Blood consists of a fluid, plasma, in which a variety of components including red blood cells are suspended. Stedman's Medical Dictionary, 26th edition, page 214 (1995). A particularly prevalent component of red blood cells is the protein hemoglobin, which is specifically responsible for the transport of oxygen from the lungs to other tissues in the body.
Because of the great demand for blood in the United States and throughout the world, intense research efforts have been directed towards the development of hemoglobin-based blood substitutes. Such substitutes have been produced with a variety of modifications of purified hemoglobin in order to prevent toxicity or otherwise enhance the properties of the molecule, with one particularly important class of modification accomplished using molecules of the polyethylene glycol (PEG) family that have been activated (aPEGs) so as to be capable of chemically modifying proteins. Such PEGylation is important because it favorably alters the activity, solubility, circulating half-life in vivo, toxicity and immunogenicity of hemoglobins. See, generally, Delgado et al. (1992) Crit. Rev. Ther. Drug Carrier Sys. 9:249–304; Greenwald et al. (2000) Crit. Rev. Ther. Drug Carrier Sys. 17: 101–161.
The literature reveals that hemoglobin has been PEGylated using a variety of aPEG compounds. One aPEG used to modify hemoglobin is the activated PEG molecule polyoxyethylene (α-carboxymethyl, ω-carboxymethoxypolyoxyethylene) (POE), which is reacted with pyridoxylated hemoglobin to obtain pyridoxylated hemoglobin polyoxyethylene (PHP). Talarico et al. (1999) Biochem. et Biophys. Acta 1476:53–65, herein incorporated by reference. POE-modified hemoglobins are of particular interest because they contain cellular proteins that enhance their functionality as scavengers of nitric oxide (NO), a compound implicated in a variety of diseases. Thus there is great potential use of such PHPs as therapeutic agents for the treatment of diseases linked to the presence of excess NO including, for example, systemic inflammatory response syndrome (SIRS), a critical medical illness which is recalcitrant to existing therapies. Privalle et al. (2000) Free Radic. Biol. Med. 28:1507–17, herein incorporated by reference.
Although PHP and other PEGylated hemoglobins have great promise in these and other therapeutic applications, there are a number of problems affecting such uses, notably the presence of contaminating compounds that affect their safety or efficacy. Dangerous viruses such as the HIV/AIDS virus or hepatitis viruses, for example, often contaminate the whole blood used to prepare the hemoglobin fraction used in PEGylation, and must be removed at some point during preparation of the final PEGylated product by filtration or other means. Additionally, bioburden such as bacteria can be a source of contamination of either the hemoglobin or raw materials such as an aPEG, as can endotoxins, the complex cell-wall lipopolysaccharide (LPS) macromolecules of gram-negative bacteria that can cause fever, diarrhea, hemorrhagic shock, and other tissue damage. Stedman's Medical Dictionary, 26th edition, page 572 (1995).
Bioburden and endotoxins are a particularly problematic source of contamination because of the lack of adequate methods for removing them from the aPEG raw material used in PEGylation. As discussed previously, aPEG molecules are activated molecules of the polyethylene glycol family. Many such activated molecules are labile in water, and as a result are typically added to the hemoglobin fraction in a powdered form, rather than in solution. Although aPEGs in this form are relatively stable, as powders they cannot be processed for the removal of contaminants using the methods available for dissolved aPEGs, such as filtration techniques. Although these contaminants can be removed post-PEGylation, such removal has undesirable consequences. For example, while endotoxins can be removed from PHP by chromatography, such chromatography results in undesirable changes to the protein composition of the PHP.
Thus there is a long-felt need for a method of purifying aPEGs for use with peptide molecules and polypeptides such as hemoglobin. Furthermore, there is a long-felt need for methods of using such purified dissolved aPEGs in the PEGylation of peptides and polypeptides such as hemoglobin, wherein the solvent in which the aPEG is dissolved does not result in the substantial denaturation or degradation of the pepetide or polypeptide such as hemoglobin.